Poehner W J, Fossarello M, Rapoport A L, Aleman T S, Cideciyan A V, Jacobson S G, Wright A F, Danciger M, Farber D B
Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA 90095-7008, USA.
Mol Vis. 2000 Oct 30;6:192-8.
We have been engaged in an ongoing study to screen candidate genes for mutations in small families with various forms of autosomal recessive retinal dystrophy. Here we report the screening of a cohort of 14 families from Sardinia for mutations in the genes encoding the alpha- and beta-subunits of cGMP-phosphodiesterase and RPE65 (PDE6A, PDE6B, and RPE65).
Haplotype analysis was performed on each family using simple sequence repeat markers closely flanking or within each of the three gene candidates. For families in which a gene could not be ruled out from segregating with disease, exons of the gene from proband DNAs were screened for mutations by SSCPE (single strand conformation polymorphism electrophoresis). All variants found by SSCPE were sequenced directly.
By haplotype analysis, 6/14, 11/14, and 4/13 families were ruled out for PDE6A, PDE6B, and RPE65, respectively. A few variants were found in the proband DNAs of the remaining families, but only one was significant: a 20 bp deletion in exon 4 of RPE65. The deletion co-segregated with disease in one family and caused a frame shift that produces a stop codon downstream. It was absent from the other Sardinian families that we tested, and from Sardinian and North American controls. Results of studies of phenotype in homozygotes and heterozygotes in this Sardinian family are compared with those from a non-Sardinian family recently reported to have the same RPE65 mutation.
This RPE65 mutation, which appears to be quite restricted in its occurrence in Sardinia, leads to childhood onset severe retinal dystrophy or Leber congenital amaurosis. Affecteds of the other 13 plus two additional families were diagnosed with arRP. This family lived in an area of Sardinia where none of the others lived suggesting different ancestral origins.
我们一直在进行一项持续性研究，以筛查各种常染色体隐性视网膜营养不良的小家族中的候选基因突变。在此，我们报告对来自撒丁岛的14个家族进行队列研究，以筛查编码环磷酸鸟苷磷酸二酯酶α和β亚基以及RPE65（PDE6A、PDE6B和RPE65）的基因突变。
对每个家族使用紧密位于三个候选基因侧翼或基因内部的简单序列重复标记进行单倍型分析。对于不能排除与疾病共分离的基因的家族，通过单链构象多态性电泳（SSCPE）筛查先证者DNA中该基因的外显子以寻找突变。通过SSCPE发现的所有变异均直接进行测序。
通过单倍型分析，分别排除了6/14、11/14和4/13的家族与PDE6A、PDE6B和RPE65相关。在其余家族的先证者DNA中发现了一些变异，但只有一个具有显著意义：RPE65外显子4中的20bp缺失。该缺失在一个家族中与疾病共分离，并导致移码，在下游产生一个终止密码子。在我们检测的其他撒丁岛家族以及撒丁岛和北美对照中均未发现该缺失。将这个撒丁岛家族中纯合子和杂合子的表型研究结果与最近报道的具有相同RPE65突变的非撒丁岛家族的结果进行了比较。
这种RPE65突变在撒丁岛的出现似乎非常有限，会导致儿童期发病的严重视网膜营养不良或莱伯先天性黑蒙。其他13个家族以及另外两个家族的患者被诊断为常染色体隐性视网膜色素变性。这个家族生活在撒丁岛的一个其他家族都不住的地区，表明其祖先起源不同。
